Bacteria have been used to produce a wide range of commercial products. For example, many Streptomyces strains and Bacillus strains have been used to produce antibiotics; Pseudomonas denitrificans and many Propionibacterium strains have been used to produce vitamin B12; some other bacteria have been used to produce vitamin Riboflavin; Brevibacterium flavum and Corynebacterium glutamicum have been used to produce lysine and glutamic acid, respectively, as food additives; other bacteria have been used to produce other amino acids used as food additives; Alcaligenes eutrophas has been used to produce biodegradable microbial plastics; and many Acetobacter and Gluconobacter strains have been used to produce vinegar. More recently, it has become common for bacteria, such as Escherichia coli (E. coli), to be genetically engineered and used as host cells for the production of biological reagents, such as proteins and nucleic acids, in laboratory as well as industrial settings. The pharmaceutical industry supports several examples of successful products which are human proteins which are manufactured in E. coli cultures cultivated in a fermenter.
It is not an uncommon occurrence for normal bacterial proteins to adversely affect the production or the purification of a desired protein product from an engineered bacteria. For example, when E. coli bacteria are used as host cells to generate in large quantity of a desired product encoded by a gene that is introduced into the host cells by a plasmid, certain normal E. coli gene products can interfere with the introduction and maintenance of plasmid DNA. More significantly, because of the economies of bacterial culture in making proteins in bacteria, often the cost of purification of a recombinant protein can be more than the cost of production, and some of the natural proteins produced by the bacterial host are sensitive purification problems. Many bacterial strains produce toxins that must be purified away from the target protein being produced and some strains can produce, by coincidence, native proteins that are close in size to the target protein, thereby making size separation not available for the purification process.
Also, however, the genome of a bacteria used in a fermenter to produce a recombinant protein includes many unnecessary genes. A bacteria living in a natural environment has many condition responsive genes to provide mechanisms for surviving difficult environmental conditions of temperature, stress or lack of food source. Bacteria living in a fermentation tank do not have these problems and hence do not require these condition responsive, genes. The bacterial host spends metabolic energy each multiplication cycle replicating these genes. Thus the unnecessary genes and the unneeded proteins, produced by a bacterial host used for production of recombinant protein, simply represent lack of efficiencies in the system that could be improved upon.
It is not terribly difficult to make deletions in the genome of a microorganism. One can perform random deletion studies in organisms by simply deleting genomic regions to study what traits of the organism are lost by the deleted genes. It is more difficult, however, to make targeted deletions of specific regions of genomic DNA and more difficult still if one of the objectives of the method is to leave no inserted DNA, here termed a “scar,” behind in the organism after the deletion. If regions of inserted DNA, i.e. scars, are left behind after a genomic deletion procedure, those regions can be the locations for unwanted recombination events that could excise from the genome regions that are desirable or engender genome rearrangements. Since in building a series of multiple deletions, scars left behind in previous steps could become artifactual targets for succeeding steps of deletion. This is especially so when the method is used repeatedly to generate a series of deletions from the genome. In other words, the organism becomes by the deletion process genetically unstable if inserted DNA is left behind.
Another attribute of interest in bacterial strains of laboratory and industrial use is what is referred to as transformation competence. It is desirable to introduce exogenous DNA into bacteria in culture so that the bacteria will then stably maintain and reproduce the introduced DNA. Bacterial strains vary in their ability to take up and maintain foreign DNA. This characteristic is their transformation competence.
Certain treatments and culture conditions can affect transformation competence. In general, E. coli cells grown in normal growth medium, unlike certain bacilli, do not take up exogenous DNA. It was discovered by Mandel and Higa (J Mol Biol 53: 159-162 (1970)) that treatment of E. coli with calcium chloride allowed E. coli to take up DNA from bacteriophage lambda. Cohen et al (Proc Nat Acad Sci 69: 2110 (1972)) observed that E. coli could be transformed by this method with plasmid DNA. The state of E. coli induced by this treatment, in which they become able to take up DNA became known as “transformation competence” and the cells as “transformation competent cells” or more simply, as “competent cell”. Under the Mandel and Higa conditions, the yield of transformants is typically in the region of 105 to 106 transformants per microgram of plasmid DNA.
Since the early observations of Mandel and Higa, many variations of the procedure have been tried in attempts to increase the efficiency of the procedure, in order to maximize the number of transformants per unit input of DNA, and to improve the reproducibility of the procedure notably by Hanahan and coworkers (J Mol Biol 166: 557-580). The method of Hanahan is used widely to induce competence. Typically, the yield of transformants using the described method is 105 to 108 transformants per microgram of plasmid DNA.
Generally, researchers have found that the transformation efficiency of competent cells is very variable and it has proven difficult to achieve reproducible results (discussed by Hengen, P. N., Trends in Biochemical Sciences 19:426-427 (1994) and Trends in Biochemical Sciences 21:75-76 (1996)). It has been found that growth at lower than normal temperature (18° C. to 32° C.) produces higher competence (U.S. Pat. No. 4,981,797 and Inoue H et al, Gene 96:23-28 (1990). There is a need for procedures that reproducibly produce competent cells with high transformation efficiency.
Transformation competence is a variable from strain to strain of bacterial hosts. Hanahan found that strain MM294 transformed readily, and developed a derivative strain, DH1, from which many of the strains in use today have been derived. Introduction of the deoR mutation (in strain DH5) appeared to increase the transformation efficiency (Hanahan Methods in Enzymology 204:63-113 (1991) and U.S. Pat. No. 4,851,348) though the molecular mechanism of the improvement is still unknown. DH5 has been used extensively for transformation. Commercial strains have also been derived from strain MM294, via strain JM109 and strain AG1. Strain AG1 contains one or more additional uncharacterized mutations that increases transformation efficiency (Bullock et al, Biotechniques 5:376 (1987)). The commonly used strain DH10b, which also contains the deoR mutation, was 30 times better than DH1 in transformation with 66 kb plasmid DNA. Its pedigree is different from DH1 and DH5, though they share common ancestors. All of the commonly used strains have undergone numerous mutational and recombinational treatments) and are very distant from wild-type E. coli (Bachman, B Derivation and Genotypes of Some Mutants and Derivatives of E. coli K12, Vol 2, chapter 133, 2460-2488, in: Escherichia coli and Salmonella, Neidhardt, F, editor, 2nd Edition, ASM Press, Washington (1996)). There is a need for better characterized strains for transformation work that are nearer to wild-type.
One of the most effective methods of introducing DNA into E. coli consists of subjecting the cells to an electric field while they are suspended in a defined medium of low ionic strength containing the DNA it is desired to introduce. This process is known as “electroporation”. The transformation efficiency, that is the number of cells that are stably transformed by a unit measure of DNA, may be higher than in competent cells produced by other methods. A high and reproducible efficiency of transformation is desirable because it facilitates or in some cases makes possible cloning of rare and valuable DNA molecules, particularly those available in only small amounts. In particular electroporation is the preferred process for preparing cells for use in cloning bacterial artificial chromosomes (“BACs”) and other large DNA vectors commonly used in genomic DNA sequencing.
In order to be competent for transformation using the electroporation method, known as “electroporation competent” or “electro-competent”, the E. coli cells need to be grown under certain specific conditions and subjected to certain specific treatments. Methods for inducing electroporation competence are described in Dower et al (Nucleic Acid Research 16:6127-6145 (1988)), Calvin and Hanahan (J Bacteriol 170:2796-2801 (1988)) and U.S. Pat. No. 4,910,140 and U.S. Pat. No. 5,186,800. These publications also discuss the many factors that affect the competence and transformation efficiency of E. coli. 
However, the methods described in the literature for preparing electroporation competent cells are not ideal. The efficiency of transformation is variable depending upon the strain of bacteria used, the conditions used to grow the bacteria and the conditions used to expose the bacteria to the electric field. In particular, only two growth media have been disclosed for the preparation of competent cells: Luria-Bertani broth, commonly known as ‘LB broth’ (Miller J H (1972) Experiments in Molecular Biology, Cold Spring Harbor Laboratory, New York) and SOB medium, described by Hanahan (J Mol Biol 166: 557-580). The composition of LB (per liter of medium) is Bacto Tryptone 10 g, Bacto Yeast Extract 5 g and sodium chloride 10 g. The composition of SOB (per liter of medium) is Tryptone 20 g, Yeast Extract 5 g, sodium chloride 0.5 g, magnesium sulphate 2.4 g and potassium chloride 0.126 g. Neither medium has any additional carbon source, such as glucose or other carbohydrate, or glycerol, or additional pH buffering component such as phosphate or non-metabolisable compounds such as MOPS or Tris. The amino acid compounds in the tryptone and yeast extract serve as both nitrogen and carbon sources.
The stage of the growth cycle at which cells are harvested has been described as critical to success, and there is a narrow window in the cell concentration at harvest, above which, or below which, the transformation efficiency drops off rapidly (Calvin and Hanawalt (1988); Dower et al (1988), supra). In LB medium, the optimal stage of the growth cycle occurs at a cell concentration corresponding to an optical density of 0.6. This is a relatively low cell density, since E. coli can grow to cell densities many times higher than this under appropriate fermentation conditions, well known to those trained in the art of fermentation. As a result, the process of preparing electroporation competent cells is wasteful of expensive growth media, fermentor capacity and time, and operator time, compared with a process that could use cells grown to higher cell densities. It also requires more expensive downstream processing equipment and time, since the competent cells need to be recovered from larger volumes of dilute suspension. There is a need for better methods for preparing electroporation competent cells, in particular it would be economically beneficial to have a process in which cells can be grown to much higher cell densities without losing transformation efficiency.
As further evidence of the difficulties scientists face in making competent cells, a market has developed for pre-prepared, frozen competent E. coli cells that exhibit a higher, more consistent and reproducible efficiency of transformation. In producing commercial competent cells, manufacturers have resorted to several conditions to improve their processes. U.S. Pat. No. 4,981,797 discloses an improved process operating at temperatures between 18° C. and 32° C., but nevertheless, cells need to be harvested at low cell concentrations. U.S. Pat. No. 6,040,184, U.S. Pat. No. 6,338,965 and WO 0022147 disclose the addition of sorbitol or other sugars to improve the transformation efficiency. WO 0109362 discloses a procedure to select variant bacteria that are more tolerant of the killing effect of the high field electrical pulse used in the electroporation procedure.